Calf thymus DNA (CT-DNA), pBR322 DNA, ethidium bromide (EB) solution, and albumin human serum (HSA) were purchased from Solarbio (Beijing, China). DMEM medium, fetal bovine serum, penicillin/streptomycin, and PBS buffer solution used in cell culture were purchased from Sangon Biotech (Shanghai, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Human cervical cancer cells (HeLa cells) were provided from Shanxi Key Laboratory of Pharmaceutical Biotechnology (Taiyuan, China). Other chemicals and solvents used were purchased from locally available suppliers.

Synthesis of the [Fe(II)(DPA-Bpy)(NCCH₃)](OTf)₂ complex: The synthesis of the pentadendate ligand, N,N-bis(2-pyridinylmethyl)-2,20-bipyridine-6-methanamine (DPA-Bpy) was achieved by our previous method (Radaram et al., 2011; Singh et al., 2012). The [Fe(DPA-Bpy)(NCCH₃)](OTf)₂ complex was prepared with a modified method. The reaction of Fe(OTf)₂ (145 mg, 0.40 mmol) with DPA-Bpy (150 mg, 0.40 mmol) in CH₃CN (30 ml) at room temperature for overnight results in a reddish cloudy solution. The precipitate was filtrated and washed with Et₂O and then dried under vacuum to yield [Fe(DPA-Bpy)(NCCH₃)](OTf)₂ as a dark red powder (Scheme 1). Yield: 270 mg, 89%. ¹H NMR (CD₃CN): 9.10 (d, 1H, J = 5.4 Hz, bipy-H13), 8.57 (d, 1H, J = 8.0 Hz, bipy-H10), 8.32 (d, 1H, J = 7.9 Hz bipy-H9), 8.29 (td, 1H, J = 8.0, 1.4 Hz bipy-H11), 7.87 (t, 1H, J = 8.0 Hz, bipyH-12), 7.82 (td, 1H, J = 7.9, 1.4 Hz, bipy-H8), 7.71 (td, 2H, J = 7.9 1.4 Hz, pyr-H3), 7.42 (d, 2H, J = 7.9 Hz, pyr-H2), 7.26 (d, 1H, J = 7.9 Hz, bipy-H7), 7.0 (t, 2H, J = 6.6 Hz, pyr-H4), 6.83 (d, 2H, J = 5.6 Hz, pyr-H5), 4.95 (d, 2H, J = 15.9 Hz, pyrCH₂), 4.86 (d, 2H, J = 15.9 Hz, pyrCH₂), 4.91 (dd, 4H, J = 15.9 Hz, pyrCH₂), and 4.75 (s, 2H, bipyCH₂). ESI-MS: m/z ⁺ 572.30 (Calcd m/z ⁺ for [Fe(DPA-Bpy)(OTf)]⁺, 572.07). Anal. Calcd for C₂₇H₂₄F₆FeN₆O₆S₂: C, 42.53; H, 3.17; N, 11.02. Found: C, 42.40; H, 3.09; N, 10.79. NMR and MS spectra for the complex are submitted as Supplementary Material.

¹H NMR spectra were measured by the Bruker 500 MHz NMR spectrometer. Elemental analysis of the [Fe(DPA-Bpy)(NCCH₃)](OTf)₂ complex was conducted by Atlantic Microlab, Inc., Atlanta, Georgia. The mass spectra were obtained by the Thermo Q Exactive field and Bruker Ultraflex MALDI-TOF MS spectrometer. UV–Vis spectra were recorded on a Thermo Evolution-220 spectrometer. Fluorescence spectra were recorded on a FluoroMax-4 spectrofluorometer.

Fluorescence spectroscopy was measured to analyze the interaction of the Fe complex with CT-DNA. The concentration of the CT-DNA stock solution was calculated by determining the absorption at 260 nm. Then, the CT DNA-EB solution was prepared by mixing 2.5 μM EB and 170 μM CT-DNA in a 10 mM Tris buffer (pH 7.4). Finally, the titrations were conducted by the addition of the Fe complex (0–20 μM) to the CT-DNA-EB solution step by step until a plateau in the fluorescence intensity was reached. The sample solution was stirred gently at room temperature, and the fluorescence spectra were collected in the range of 530–750 nm using 1-cm quartz every 5 min. The excitation wavelength was 520 nm, and slit width was set to 5 nm.

The interaction of the Fe complex with plasmid DNA was analyzed by agarose gel electrophoresis. The supercoiled pBR322 DNA alone was used as the control and then pBR322 DNA (2 μg) and the Fe complex (10–50 μM) were mixed and the buffer solution was added to a total volume of 20 μl. The mixed solutions were protected from light and incubated for 30 min. Finally, the solutions were electrophoresed at 70 V through 1% agarose gel immersed in 1× TBE buffer for 40 min.

The binding of the Fe complex with HSA was explored by the fluorescence quenching method and MALDI-TOF MS spectra. Different concentrations of the complex (0–50 μM) were added to HSA (10 μM) in 20 mM PB buffer (pH 7.4), and then the fluorescence spectra were recorded in a range of 290–520 nm with 280 nm as the excitation wavelength. Both the slit-width for excitation and emission were set to 5 nm. 2,5-dihydroxybenzoic acid (DHB) was used as the matrix for MALDI-TOF MS spectral measurement. HSA (5 mg/ml) and the Fe complex (the ratio was 1:5) were mixed in 20 mM PB buffer (pH 7.4) and kept at 4°C in the refrigerator overnight. The mass spectra were recorded from 30,000 to 150,000.

Crystallization of HSA was performed with the hanging-drop vapor diffusion method at 18°C. The drops consisted of 1.5 μl of the HSA solution and 1.5 μl of reservoir solution. The crystallization reservoir contains 25% (w/v) polyethylene glycol 3,350, 5% glycerol, 5% DMSO, and 50 mM potassium phosphate (pH 7.5). The native crystals were soaked in a solution containing 5 mM Fe complex, 25% (w/v) PEG 3350, and 25 mM potassium phosphate buffer for 5 h. The X-ray diffraction dataset to a maximum resolution of 2.4 Å was collected at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). The data were processed, merged, and scaled with the HKL-3000 (Minor et al., 2010). The structure was determined by molecular replacement using the Phaser program in the Phenix program package (McCoy et al., 2007). The coordinates of HSA and [RuCl₅(indazol)]^2- complex adduct (PDB ID: 5GIY) were used as an initial model (Zhang et al., 2014). The structure was rebuilt using COOT (Emsley and Cowtan, 2004), and the structural refinement was conducted using the program Phenix (Adams et al., 2002). The model geometry was verified using MolProbity (Chen et al., 2010), and the structural figures were drawn using PyMOL (DeLano, 2002).

The cytotoxicity of the Fe complex was evaluated against HeLa cells using CCK-8 assay. HeLa cells were cultured with a DMEM adding 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37°C under 5% CO₂ atmosphere. The cells were seeded on a 96-well plate with a density of 5 × 10⁴ cells/ml. After they were incubated for 12 h, the cultured cells were treated with the culture medium containing different concentrations of the Fe complex (0, 0.5, 1, 2, 5, and 10 μM) and incubated for another 24 h. Then, 100 μl CCK-8 solution (1 mg/ml) diluted with the cell culture medium was added to each well, and the plate was further incubated for 3 h. The absorbance was recorded at 450 nm using a SpectraMax iD5 microplate reader. Each experiment was repeated at least three times. The IC₅₀ values of the complex against the HeLa cell were calculated by plotting the viability versus concentration on a logarithmic scale.

The effects of the complex on the HeLa cell cycle and apoptosis were analyzed by the flow cytometric technique. Cell cycle arrest was performed by staining the cell with PI. Apoptosis assays were evaluated using Annexin V-FITC as an apoptosis detection kit.

The Fe complex structure was confirmed from different spectroscopy techniques including ¹H–NMR, elemental analysis, ESI–MS, and UV–Vis as described in the experimental section and supplementary information (Supplementary Figures S1–S3). At room temperature, the ¹H–NMR spectrum in acetonitrile indicates a diamagnetic Fe complex. The ¹H–NMR spectrum of the complex (Supplementary Figure S1) shows the two pyridine groups with the same NMR features, suggesting the presence of C _s symmetry in a solution with a plane containing the tertiary amine and bipyridine group. The DPA-Bpy is a strong-field ligand; it stabilizes and forms the low spin iron (II) complex [Fe(II)(DPA-Bpy)(NCCH₃)](OTf)₂. The Fe complex is highly soluble in water, while DPA-Bpy ligand is insoluble.

The mass spectrometry appearance of the molecular ion at 572 provided further evidence for the metal complex in positive mode corresponding to [Fe(DPA-Bpy)(OTf)]⁺ (Supplementary Figure S2). The central Fe atom has six-coordinated structures with one DPA-Bpy ligand and one solvated ligand, similar to that of [Ru(DPA-Bpy)Cl]Cl and [Co(DPA-Bpy)Cl]Cl (Radaram et al., 2011; Singh et al., 2012). As shown in Supplementary Figure S3, the UV–Vis spectrum of the complex in acetonitrile shows two obvious absorption bands at 350–600 nm from charge transfer transitions between the metal ion and ligand.

Since the potential and vital biological targets of metal complexes are DNA and proteins, the interactions of metal complexes with DNA/protein have attracted great interest in determining the mode of binding. The binding analyses for metal complexes and DNA is the key to understanding the mechanism of their pharmacological action (Murray et al., 2018; Shao et al., 2021). Interactions of the Fe complex with CT-DNA were studied by fluorescence spectroscopy and agarose gel electrophoresis.

Fluorescence spectroscopy is a sensitive technique to analyze the binding pattern and strength of the complex with CT-DNA using EB as a fluorescence probe. The strong emission intensity at 598 nm for the CT-DNA-EB adduct was observed when the EB aromatic ring is inserted into the base pair of DNA double helix. However, the fluorescence intensity decreases obviously after the addition of the Fe complex, and due to that, the complex replaces EB in the EB-DNA system (Figure 1). The binding constant (K _b) and the number of average binding sites (n) are calculated according to the Stern–Volmer Eqs 1, 2 (Seyedi et al., 2020; Shao et al., 2021). F 0 F = 1 + K Q τ 0 [ Q ] = 1 + K S V [ Q ] . (1) log ( F 0 - F ) /F  =  log  K b + n log [ Q ]   . (2)

To better understand the thermodynamics of the reaction between the Fe complex and DNA, the changes of standard entropy (∆S), enthalpy (∆H), and Gibbs free energy (∆G) are further evaluated at three different temperatures according to Eq. 3. The results are listed in Table 1. The K _b is 5.00–5.96 × 10⁵ M⁻¹, and the average binding-site number is about 1.2. The positive value of ∆S (and ∆H near to 0) suggested that electrostatic interactions exist between the complex and CT-DNA (Yang et al., 2011; Heydari and Mansouri-Torshizi, 2016; Seyedi et al., 2020; Shao et al., 2021). The negative value of ∆G shows the spontaneity of the reaction, and the negative value of ∆H reveals that the interaction of the Fe complex with DNA is exothermic. This is in agreement with decrease in the K _b values upon increasing the temperature (Table 1). Δ G= Δ H   -   Δ S   =   - RT ln  K b   . (3)

In order to investigate the effect of the Fe complex on the configuration of DNA, the technique of agarose gel electrophoresis was used, and the results are shown in Figure 2. The plasmid pBR322 DNA with different configurations moves at different velocities in the electric field. The closed superhelical form (CC) migrates faster in the electrophoresis process, while the open-circular form (OC) moves slower because of the single-strand breakage (Zhou et al., 2009). There is just the CC form for DNA alone in Lane 1 for control. As the concentration of the Fe complex increased, more OC forms of DNA were observed in Lanes 2–4. The 10 mM Fe complex led to an obvious interaction of DNA, while almost no damage was observed for the ligand alone at the same concentration. It indicated that the coordination of Fe with the DPA-Bpy ligand is necessary to induce DNA cleavage, in which it is possible through a free radical mechanism such as bleomycin Fe complex (Ma et al., 2009; Myers et al., 2013; Murray et al., 2018).

Human serum albumin (HSA) is the most abundant protein in plasma and is the main carrier of endogenous and exogenous ligands, including fatty acids (FAs), nucleic acids, hormones, metals, toxins, and drugs. Thus, HSA plays a relevant role as a drug carrier by influencing their pharmacokinetics and pharmacodynamics (Ishima et al., 2012; Belinskaia et al., 2021; De Simone et al., 2021). Furthermore, the fluorescence spectroscopy and MALDI-TOF MS spectra were used to study the binding mode and interaction between the synthesized complex and HSA.

The HSA is intrinsically fluorescence active which comes from Trp, Tyr, and Phe residues. As shown in Figure 3, the fluorescence is very sensitive to its microenvironment, where the fluorescence quenching of HSA is observed with increasing concentrations of the Fe complex. It indicated that the molecular interactions and binding of the Fe complex with serum protein affects the chromogenic groups.

The Stern–Volmer Eq. 2 is used to analyze the fluorescence quenching data at three temperatures, and the corresponding calculated results are listed in Table 2. The K _b is 1.34–3.09 × 10⁴ M⁻¹, and the average binding-site number is close to 1. The calculated ∆H, ∆S, and ∆G are shown in Table 2. The negative values of ∆S and ∆H are frequently taken as evidence for the hydrogen bond and van der Waals power. The negative value of ∆G shows the spontaneity of the reaction. The increase in the K _b values upon increasing the temperature and the positive value of ∆H reveals that the binding process of the Fe complex with HSA is endothermic (Zhou et al., 2009; Yang et al., 2011; Shao et al., 2021).

The HSA molecule is a single-chain protein which contains 585 amino acid residues in a heart-like shape, and the three domains in HSA provide binding sites for a wide variety of endogenous ligands (Ghuman et al., 2005; Xie et al., 2021). To examine the number of the Fe complex bound to HSA in solution, the native and Fe complex–treated HSA were analyzed by MALDI-TOF MS. The MS spectra for native HSA and Fe-HSA complex adduct are shown in Figure 4; the spectra in the DHB matrix revealed that the m/z of native HSA was 65,694 and that of the HSA incubated with the Fe complex was 66,110. The difference of the m/z value is 416, which is just close to the molecular fragment mass (423.3) of one [Fe (DPA-Bpy)] complex.

HSA plays a key role in regulating the distribution of many natural and artificial small molecules. The interaction of the complex with HSA may provide information for understanding their biodistribution, metabolic process, and pharmacological mechanisms of complexes (Zhu et al., 2008; Yamaguchi et al., 2010; Wang et al., 2013; Qi et al., 2016; Ito et al., 2020). To further investigate the binding mode of the Fe complex to HSA, the crystal structure of HSA and the Fe complex adduct was determined by using the X-ray diffraction method. The structure was refined to 2.4 Å resolution, and the coordinate has been deposited to the Protein Data Bank (PDB ID: 7WLF). The final refinement statistics are summarized in Table 3.

The overall structure is shown in Figure 5, and the crystal structure indicates that HSA has three typical domains (I–III). The refined electron density map clearly revealed that one Fe complex molecule is coordinated with His288 and binding in site between the interface of domain I and domain II. The Fe center is coordinated with 1 N atom of His288 and 5 N atoms of DPA-bpy ligand. The six bond lengths between Fe and N atoms are in the range 1.80–2.18 Å, as shown in Figure 6. The binding site is located near the center region of the heart structure of HSA and exposed to the surface of the protein. This site is just a little above the well-known warfarin binding sites (called as drug binding site 1 in domain II, PDB: 2BXD) of HSA (Ghuman et al., 2005; Zhu et al., 2008).

The Fe complex is located at the pocket delimited by residues including Glu153, Phe156, Phe157, Lys159, Arg160, Lys281, Glu285, and Glu292. The occupancy of the metal center is 0.46, and the B-factors for the Fe atom is 64.87. The binding site is different with the reported HSA complex structures of other Ru complexes, Cu(II) and Fe(III) complexes. There are two binding sites for the [RuCl₅(ind)]^2- complex (Zhang et al., 2014); the Ru atoms coordinated with His146 in the IB subdomain and His242 and Lys199 residues in the IIA subdomain of HSA. For a Cu(II) complex with tridentate (E)-N'-(5-bromo-2-hydroxybenzylidene) benzohydrazide Schiff base ligand (Gou et al., 2016b) and Fe(III) complex containing the tridentate 2-hydroxy-1-naphthaldehyde thiosemicarbazone Schiff base ligand (Qi et al., 2016), the central metal ions were coordinated with His242 and Lys199 residues of HSA by replacing the other two ligands of the complexes. In addition, seven palmitic acid (PA) molecules are present in the subdomains of I–III and in the border region between domains II and III.

Antitumor and antiproliferative activities of the first series of transition metal complexes containing different ligands have been reported (Chen et al., 2017; Monro et al., 2019; Icsel et al., 2020; Loginova et al., 2020; Odachowski et al., 2020). The iron as a common, abundant, and important element has attracted great attention for its cytotoxicity and genotoxicity. Though the mechanism of action for Fe complexes is complicated, studies on the interactions of metal complexes with bio-macromolecules possibly shed light on the mechanism of activity and metabolism. Cytotoxicity of the Fe complex and Fe complex HSA adduct was evaluated against HeLa cells using the CCK-8 method. Figure 7 showed the effect of the Fe complex and its HSA complex adduct on the cell viability of HeLa cells and normal liver cell HL-7702, where the mortality of HeLa cells increased as the concentrations of the Fe complex and its HSA complex adduct increased. The complex had a certain inhibitory effect on the growth of the cancer cells, and the IC₅₀ of the Fe complex and its HSA complex adduct was 1.18 and 0.82 μM, respectively, for the tested tumor cell, while the Fe complex and its HSA complex adduct had a less inhibitory effect on the normal liver cell HL-7702 at similar concentration ranges.

Furthermore, the flow cytometric data were analyzed to examine the effects of the Fe complex on HeLa cell cycle distribution. As shown in Figure 8A, the percentage of G1 phase for the negative control is 66.0%, and it decreases to 56.0 and 48.2% at concentrations of 0.5 and 0.8 µM, respectively. The percentages of cells in the S phase increased from 18.6 to 29.1% and 34.8% at the same concentrations. These results indicated that the Fe complex induced a dose-dependent S phase arrest of HeLa cells. It is similar to that of the Fe complex with heterocyclic thiosemicarbazone ligands (Gou et al., 2016a). For the Fe complex HSA adduct (Figure 8B), the percentage of G1 phase decreases from 62.0 to 46.8% and 43.3% at concentrations of 0.5 and 0.8 µM, respectively. The percentages of cells in the S phase increased from 28.1 to 42.8% and 45.7% at the similar concentrations. The effects of the Fe complex and the HSA Fe complex adduct on the apoptosis of HeLa cells were shown in Figure 9. The Fe complex HSA adduct showed a higher inhibitory effect than that of the Fe complex alone, which is consistent with their cytotoxicity.